Process of producing an antibiotic of the tetracycline series



United States Pate'nt PROCESS or PRODUCING ANANTIBIOTIC on THE TETRACYCLINEJSERIES Jerry Robert Daniel McCormick-New City, Nancy Hazlett Arnold-and Ursula .Hirsch,.Pearl.River, and Philip .Andrew .Miller, .New City, .N.Y., and Newell Oscar Sjolander, Saddle River, N.,I,, a ssigprs to. American Qyanamid Company, New York, N.Y., a.corporati0n .of Maine No Drawing. -0riginal application June20, 1958, Ser. No. 743,424. Divided and this --application Mar. .12, 1959, Ser. No. 798,857

Claims. (c1. 1195- 80 This application is a division of ourcopending application Serial No. 743,424, filed'June 20,.1958.

This invention relates to a novel .process of producing an antibiotic of the .tetracyclineseries and more particularly is concerned withanimprqved process .of fermenting microorganismsofthegenusStreptomyces which produce such. tetracycline-antibiotics in ,the presence of a 5a(11a)-dehydrotetracyclines [see also J.A.C.S., .80, 55.72

(.195 8) The5a(11a)-dehydrotetracyclines are produced by certain strainsof, S. aureofaciens such as strain S1308,

, for .example, the morphological and cultural characteristics of which .are adequately described inthe aforesaid copendingapplication. {In addition, viable .cultures of S. aureofaciens,strain1S1308, as well as several variants thereof haveflbeen deposited with the American Type Culture Collection inWashington, D.C., where they have been assigned ATCC Accession. Numbers 12748-12751, inclusive. The 5a(11a)-'dehydrotetracyclines are largely biologically inactive but ht may be converted by a suitable catalytic reduction process. to the well-known, broad-spectrum antibioticttetracycline.

We have now discovered that when cosynthetic factor-1 is added to a-fermentation medium inoculated with S. aureojaciens strain Sl308 (ATCcwNo. 127.43.), for example, and the culturedsgrownunder standard aerobic conditions, :the amount .of chlortetracycline produced is increased from about.100.40.0. micrograms per milliliter .to more than 5,000. micrograms per milliliter. In this case, 5.a(11a) -.dehydrotetraeycline is no.1ongerproduced. Just why the addition of .cosynthet-ic factor-1 to the fermentation produces such a high concentration of chlortetracycline when that fermenation :ordinarily produces only minimal amounts of chlortetracycline is not known with certainty, and no theory is advanced with respect thereto. Itis known, however, that-CF-l is not a precursor as is shown by the fact that one microgram of CF-l when added to a suflicient quantity of S. aureofaciens ATCC No. 12748 results in the .biosynthesis of up to 57 milligrams of chlortetracycline beyond that normally produced a under the same conditions.

Cosynthetic factor-1 is composed of the elements carbon, hydrogen, nitrogen and oxygen. Elementary analytical determinations of purified samples indicate elementary proportions by weight substantially as follows:

carbon, 50.33%; hydrogen, 5.00%; nitrogen, 12.18%;

oxygen (direct), 32.58%. The compound is of relatively 2' low molecular weight, 340-360. The empirical formula based on the analytical values above corresponds closely to C H N O The product is soluble in waterat pH 6 and is soluble in phenol. It is insoluble in nbutanol, acetone, ether and in water at pH 1-2.

Cosynthetic factor-1 has an Rf value of 0.07 on a paper chromatogram developed with 1:1 n-butanol-water system and an Rf value of 0.28-0.35 in a 3: 1:4 n-butanolacetic acid-water system, and an Rf value of 0.30-0.40 in pH 10 ammonia-water system.

In a fifty tube Craig counter current distribution opera tion utilizing 1:1 phenol-chloroform as the organic phase and 0.1 N HCl as the aqueous phase, CF-l appears as a single. component witha peak concentration at tube 31.

An infrared absorption spectrum of a sample of the compound as obtained from dilute hydrochloric acid solution (acid-form) when suspended in a potassium bromide pellet exhibits characteristic absorption in the infrared region of the spectrum at the following wave lengths expressed in microns: 2.84, 3.18, 3.35, 3.55, 5.91, 6.02, 6.27, 6.35, 6.49, 6.64, 6.72, 7.02, 7.18, 7.35, 7.49, 7.93, 8.16, 8.36, 8.69, 9.15, 9.30, 9.59, 9.77, 9.90, 10.11, 10.47, 11.40, 11.62, 11.81, 12.56, 12.83, 13.30 :and 14.48.

The compound as obtained from pH 7 ammonium hydroxide-solution (neutral form) when suspended in a potassium bromide pellet exhibits characteristic absorpof 1010, 580, 600and 693, respectively.

An ultraviolet absorption spectrum determined from a sample of the compound at a cencentration of 10.7 micrograms per milliliter in 0.01 N NH OI-I shows characteristic absorptionmaxima at 248 III/.4, 268 III/1., 295 m c and 419 m corresponding to extinction coefiicient values tta. of 972, 684, 349 and 1215, respectively.

The organism that we prefer to use for the production of cosynthetic factor-1, because of its ability to produce larger amounts of this novel substance is a new strain of S. aureofaciens which we have designated as W-5.

The new strain is a member of the species S. aureafaciens since it is adirect descendant of the chlortetracycline-producing strain of S. aureofaciens A377 which was isolated from the soil and is described in the United States patent to 'Duggar No. 2,482,055, and the culture is deposited at the Northern Regional Research Laboratories, Peoria, Illinois, as NRRL 2209. Mutagenic agents and selective agents used in obtaining this new strain include ultraviolet irradiation, nicotine and nitrogen mustard treatments, and phase exposure.

Viable cultures of S. aureofaciens strain WS have been deposited with the American Type Culture Collection in Washington, DC, Where this strain has 'been assigned ATCC Accession Number 13,190.

The conditions for fermentation with the new strain of S. aureofaciens are generally the same as those presently usedfor cultivating other S. aureofaciens strains. That is, the fermentation medium contains the' us'ual nutrients and essential mineral elements. suitab e substances which may provide the necessary nutrients inspores per milliliter.

suspension is used to inoculate each of the tubes containclude starch, dextrin, cane sugar, glucose, molasses, soybean meal, peanut meal, yeast, meat extracts, peptone, ammonium sulfate, urea, corn steep liquor, distillers solubles, fish meal and other conventional.substances. .The

inorganic salts that may be used include calcium carbonate, ammonium sulfate, ammonium chloride, and the various trace elements such as manganese, cobalt, zinc, copper, iron and the like.

The CF-l producing culture, such as the new strain described above, is grown aerobically in a suitable inoculum medium. The inoculum is then transferred and grown in a suitable fermentation medium and the fermen- 'tation is c-arried out at a temperature of about 22 C. to

32 C. for from 48 to 168 hours on a rotary shaker.

During the fermentation the pH is usually maintained between about 5.5 and 7.5. After the fermentation is complete, the pH of the mash usually ranges from about 6-7. The mash is then filtered without a pH adjustment. 'The CF-l may then be extracted, isolated and purified in any suitable manner.

A preferred extraction process involves filtering the mash at the prevailing pH (6-7). The pH of the filtrate is then adjusted to pH 8-9 with ammonium hydroxide and saturated with ammonium sulfate. A suitable carrier such as Arquad 16, which is along chain alkyltrimethylammonium chloride in which the alkyl groups consist of 90% hexadecyl, 6% octadecyl and 4% octadecenyl, is added and the mixture is then extracted with a.

lower alkanol, i.e., n-butanol. The n-butanol extract is then adjusted to pH 1.5-2.0 with concentrated hydrochloric acid and is back-extracted with water and the graphed on a Florisil column. Florisil is an activated magnesium silicate of approximately the following composition: MgO-l5.5% i-0.5%; SiO 84.0% 10.5%; and N-a SO -0.5%. The CF-l is eluted from the Florisil column with methyl alcohol containing a small amount of water. The CF-l rich cuts are concentrated, adjusted to pH 1 with hydrochloric acid and filtered. The filtrate 'is cooled and seeded. The resulting crystals of CF-l are collected by filtration, washed and vacuum dried;

The crude product may be purified in a standard manner by recrystallization from 0.1 HCl.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 I noculum preparation 7 t A typical medium used to grow the primary inoculum is prepared according to the following formula:

Water to 1000 milliliters.

' Eight milliliter aliquots of this medium are placed in each of a series of 8 inch test tubes and sterilized by autoclaving for 20 minutes under 15 pounds per square inch pressure. Spores of strain S. aureofaciens W-5 are washed from an agar slant with sterile distilled water to form a suspension containing approximately 60x10 A 0.33 milliliter portion of this ing an S-milliliter portion of the inoculum medium shown 1 above. The inoculated shaker tube is then incubated for 24 hours at 28 C. on a reciprocating ;at l1, 6 oscillations per minute." 7

shaker operating Sucrose grams 30.0 .Corn steep liquor milliliters 16.5 Ammonium sulfate grams 2.0 Calcium carbonate do 7.0

EXAMPLE 2 Fermentation A fermentation medium is prepared according to the following formula:

Water to 1000 milliliters.

Twenty-five milliliter'portions of the medium are placed in 250 milliliter Erlenmeyer flasks, and 0.5 milliliter of lard oil is added to each flask. The flasks containing the fermentation medium and lard oil are sterilized in an autoclave for 20 minutes under 15 pounds per square inch pressure. Following sterilization and cooling, 1 milliliter of the inoculum, prepared as in Example 1, is added to each flask and the fermentation is carried out i at 25 C., for hours, on a rotary shaker operating at revolutions per minute. The mash is assayed and found to contain one microgram per milliliter of cosynthetic factor-1.

EXAMPLE 3 Extraction from whole mash A 200 liter portion of fermentation mash prepared in a pilot tank fermentor with CF-l producing strain of S. aureofaciens W-S grown in the medium described in Example 2 and assaying one microgram of CF-l per milliliter is filtered at pH 6-7 using Hyflo Super-Cel (diatomaceous earth) in an amount approximating 15% of mash volume. The filter cake is washed with sufiicient water to give a total combined neutral filtrate volume equal to the starting mash volume. The CF-l potency of the combined neutral filtrate is about one microgram per milliliter. The combined neutral filtrate is concentrated to one-quarter starting mash volume under vacuum at 60 C. Thus the final volume of concentrated combined neutral filtrate is 50 liters.

The combined neutral filtrate concentrate is adjusted to pH 8-9 with concentrated ammonium hydroxide and then saturated with ammonium sulfate. Arquad 16 (cetyltrimethylammonium chloride) is added to this solution in the proportion of 10 milliliters of a 50% Arquad 16 solution in isopropanol to each liter of combined neutral filtrate concentrate, and the mixture stirred for one-half hour. The mixture is then combined with an equal volume of n-butanol and stirred for another onehalf hour. The resultant mixture is centrifuged in order to separate the phases. This extraction is repeated using 10 milliliters of 50% Arquad 16 per liter of combined neutral filtrate concentrate and another equal volume of n-butanol. The n-butanol extracts are then combined to yield a final volume of 100 liters- The combined nbutanol extracts are concentrated to one-quarter starting volume under vacuum at 60 C. Thus the final volume lof the concentrated combined n-butanol extracts is 25 iters;

The 25 liters of concentrated, combined n-butanol extracts are adjusted to pH 1.5-2.0 with concentrated hydrochloric acid. A 5-liter quantity of water and 200 liters of methylene chloride are added and the mixture stirred well for one-half hour. The 5-liter aqueous layer is separated from the organic phase. The pH of the 5- liter volume of back water extract is adjusted to 7.0 with ammonium'hydroxide solution before it is concentrated underrvacuumat.. 60 C. to 0.5-1.0% of 'thetoriginal height of 2 feet with this buffered Celite 545.

is mixed thoroughly with pH 8.0 butler solution in the proportions of 2 grams of Celite 545 per milliliter of buifer solution. A 9-inch diameter column is packed to a The Celite 545 containing the solution. of crude cosynthetic factor-1 (concentrate of back water extract) is packed on top of the buffered Celite 545 column. The column is developed with n-butanol buifered to pH 8.0 as above. The combined CF-l rich cuts are mixed with an equal volume or water and two volumes of methylene chloride. The mixture is shaken in a separatory funnel. The organic phase is reextracted with another one-half volume of water. The total volume of combined back water extract amounts to 8 liters which is concentrated under vacuum at 40 C. to 200 milliliters.

PURIFICATION A Florisil column is prepared in the following manner: Florisil is packed into a 3-inch diameter column to a height of 18 inches. This column is washed with 0.01 N NH OH (aqueous) then with methyl alcohol containing 10% water, and finally with 0.01 N NH OH (aqueous) again. The approximately 200 milliliters of combined back water extract concentrate is placed on top of the packed and washed Florisil column and allowed to pass through; followed by a one liter volume of 0.01 N NH OH (aqueous) and 500 milliliters of water. Cosynthetic factor-1 is then eluted from this column by means of methyl alcohol containing 10% water. The combined CF1 rich cuts are adjusted to pH 7.0 with carbon dioxide, then concentrated at less than 40 C. and under vacuum to yield approximately 100 milliliters of an aqueous solution of CF-l.

The combined CF-l rich concentrate is adjusted to pH 1.0 with hydrochloric acid, heated to 90 C. and filtered through Whatman No. 4 filter paper. The filtrate is cooled, seeded, and allowed to stand overnight at C. The resulting crystals are collected by filtration, washed first with 0.1 N hydrochloric acid solution, then with water, and finally with acetone, followed by ether, and vacuum-dried at 40 C. overnight to obtain 92 milligrams of crude product.

The crude crystals are dissolved in 0.1 N hydrochloric acid solution, heated to 90 C., and cooled in ice-Water at about 5 C. to promote crystallization. The newlyformed crystals are first washed with cold (10 C.) 0.1 N hydrochloric acid solution, then with cold water, and dried under vacuum at less than 40 C. for 3 hours to obtain 62 milligrams of the acid form of cosynthetic factor-1, melting with decomposition at 280285 C. The neutral form of the product is obtained by dissolving 5 milligrams of the acid form of CF-l in 3 milliliters of hot water by the addition of ammonium hydroxide to pH 7.5. The crystalline neutral form precipitates from solution on cooling. The crystals are washed with cold water and then vacuum dried at 40 C. The chemical analysis of CF-l and its other chemical, physical, and biological properties have already been described.

EXAMPLE 4 A standard CF-l assay is set up as follows: An inoculum of S. aureofaciens S1308 (ATCC 12748) is prepared according to the procedure shown in Example 1.

Increments of crystalline CF-l obtained as described in Example 3 are added to a fermentation medium as shown-in Example 2 except that0.1 microgram per milliliter of riboflavin is added thereto. The medium is then sterilized, inoculated with the 24-hour S. aureofaciens S1308 vegetative inoculum, incubated for hours at 25 C., and assayed for the amount of chlortetracycline present.

Ohlortetracycline Produced (Turbidimetrie Assay), meg/ml.

OF-l Added, meg/ml.

999999 DIN-COCO NQDCW-NH EXAMPLE 5 Milliliters of W-5 Neutral Filtrate per milliliter of S1308 mash Chlortetracycline Produced (Turbidirnetric Assay) By comparison of these results with those on pure CF-l in Example 4 it will be seen that the W-S mash contains 0.85 microgram of CF-l per milliliter.

EXAMPLE 6 An S. aureofaciens W-5 neutral filtrate is prepared as described in Example 5. An S. aureofaciens E504 (ATCC 13,191) fermentation is prepared and carried out as in Example 4, but substituting different volumes of the S. aureofaciens W-5 neutral filtrate for the crystalline CF-l used therein. The harvest mash assays are as follows:

7 Ohloro 6-Demethyltetracycline Produced Milliliters of W-5 Neutral Filtrate per Milliliter oi E504 Mash Turbidimetric Assay, meg/ml.

Spectrophoto metric Assay,

meg/ml.

The addition of cosynthetic factor-1 to the above S. aureofaciens fermentation results in the increased pr0- duction of 7-chloro-6-demethyltetracycline.

EXAMPLE 7 An S. aureofaciens W-5 neutral filtrate is prepared as described in Example 5. Twenty-five milliliter aliquots of the fermentation medium shown in Example 2 are placed in six 250 milliliter Erlenmeyer flasks. An inhibitor of fermentative chlorination, 2,5-dimercapto- 1,3,4-thiadiazole (DMTD), is added to two of the six flasks in the proportion of 0.10 milligram of DMTD per milliliter of fermentation medium. All six flasks are then sterilized and cooled. An S. auneojaciens S1308 fermentation is prepared and carried out as in Example 4 but substituting 0.08 milliliter of the S. aureofaciens W-5 neutral filtrate for the crystalline CF-l used therein. The harvest mash assays are as follows:

Chlortetra- Tetracycline cycline Milliliters of W-5 Neutral Filtrate DMTD, Fluoro- Spectroper Milliliter 0t S1308 Mash mg./ml. metric photo- 7 Assay, metric meg/ml. Assay,

mcgJml.

The above data show that the addition of cosynthetic factor-1 to an S. aureofaciens S1308 fermentation containing a chlorination inhibitor results in the production of tetracycline.

We claim:

1. In a process for the production of an antibiotic of 8 the .tetracycline.series which comprises growing under aerobic conditions a culture of a species of Streptomyces which produces said antibiotic in an aqueous nutrient medium, the step which comprises growing said organism in said medium invthe presence of added cosynthetic factor-1 until substantial antibacterial activity is imparted thereto. v

2. A process according to claim 1 in which the compound of the tetracycline series is chlortetracycline.

3. A process according to claim 1 in which thecompound of the tetracycline series is tetracycline. p 4. A process according to claim 1 in which the compound of the tetracycline series is 7-ch1oro-6-demethyltetracycline.

5. A process according to claim 1 in which the compound of the tetracycline series is 6-demethyltetracycline.

References Cited in thefile of this patent v UNITED STATES PATENTS 

1. IN A PROCESS FOR THE PRODUCTION OF AN ANTIBIOTIC OF THE TETRACYCLINE SERIES WHICH COMPRISES GROWING UNDER AEROBIC CONDITIONS A CULTURE OF A SPICES OF STREPTOMYCES WHICH PRODUCES SAID ANTIBIOTIC IN AN AQUEOUS NUTRIENT MEDIUM, THE STEP WHICH COMPRISES GROWING SAID ORGANISM IN SAID MEDIUM IN THE PRESENCE OF ADDED COSYNTHETIC FACTOR-1 UNTIL SUBSTANTIAL ANTIBACTERIAL ACTIVITY IN IMPARTED THERETO. 